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Related Experiment Videos

Selection of DNA clones with enhancer sequences

S Asoh1, W Lee-Kwon, M M Mouradian

  • 1Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

Proceedings of the National Academy of Sciences of the United States of America
|July 19, 1994
PubMed
Summary

Researchers developed a method to identify DNA enhancer sequences that boost gene expression. By inserting mouse DNA into a special vector, they found clones that significantly increased reporter gene activity, aiding in gene regulation studies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Gene Regulation

Background:

  • Enhancer sequences are crucial regulatory elements that control gene expression.
  • Identifying functional enhancers from genomic DNA is challenging.
  • A shuttle vector system is needed to select for DNA fragments with enhancer activity.

Purpose of the Study:

  • To develop and validate a method for selecting DNA clones containing functional enhancer sequences.
  • To identify novel mouse genomic DNA fragments that can activate gene expression.
  • To characterize the gene regulatory potential of selected DNA fragments.

Main Methods:

  • Utilized an Escherichia coli-rodent cell shuttle vector (pPyE0) lacking the polyoma enhancer region.
  • Inserted mouse genomic DNA fragments into pPyE0 adjacent to the polyoma origin of replication.

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  • Selected recombinant plasmids based on restored replication efficiency in mouse cells.
  • Transfected mouse cells with the DNA library and assessed reporter gene (chloramphenicol acetyltransferase) expression.

    • Main Results:

    • Replication of pPyE0 in mouse cells was restored by inserted DNA fragments containing putative enhancers.
    • 16 clones with DNA inserts were identified that stimulated enhancerless reporter gene expression.
    • Four clones demonstrated a 4- to 13-fold increase in reporter gene mRNA levels.
    • Sequence analysis identified 5 clones with homology to known gene regulatory regions.

    Conclusions:

    • The developed method effectively selects for DNA clones containing functional enhancer sequences.
    • Identified mouse genomic DNA fragments possess significant gene-activating potential.
    • The selected DNA sequences are likely involved in the regulation of gene expression.